The major goal of this proposal is to elucidate the molecular and genetic pathway(s) controlled by Eya1 and its interacting genes in early mammalian inner ear morphogenesis. The vertebrate inner ear develops from the otic placode, a one-cell-thick epithelium via multiple inductive processes. A large number of otic genes have been isolated recently, however, their precise functions are largely unknown and the molecular and genetic pathway(s) regulating the morphogenetic processes of inner ear development have not been established. Recently, it was found that the murine Eya1 gene is expressed during the development of the auditory system and mutations in the human EYA1 gene cause Branchio-Oto-Renal (BOR) syndrome, a congenital birth defect that accounts for as many as 2% of profoundly deaf children. However, despite the identification of the responsible gene for BOR syndrome, the developmental and molecular basis of auditory defects occurring in BOR syndrome and the identity of the steps at which Eya1 functions in early otic development are unclear. In Eya1-/- mouse embryos, the inner ear development arrests at the otic vesicle stage, indicating that Eya1 is a key gene required for early otic morphogenesis. Molecularly, Six1 but not Pax2 and Pax8 expression in the otic vesicle depends upon Eya1 function. Moreover, it was recently found that the murine Six1 is also a key gene for inner ear development and its gene product physically interacts with Eya1 in cultured cells. Thus, we hypothesize that during early otic morphogenesis, Pax8 or Pax2 regulates Eya1 and Six1 whose respective gene products interact. This grant will use a powerful genetic system to test this hypothesis. Specifically, I propose to: (1) establish the developmental and molecular mechanism(s) by which Eya1 acts in early otic development by phenotypic and molecular analyses, (2) test the functional role of Six1 and the possible interactions between Six1 and Eya1 in early otic morphogenesis in vivo, (3) test the hypothesis that the Pax-Eya-Six regulatory hierarchy is utilized during early otic development by examining the pattern of gene expression in the respective mutants and co-localization studies, (4) test whether the candidate Eya1-interacting proteins isolated from yeast two-hybrid screen function in the Eya1-Six1-regulatory pathway in early otic development. These studies will clarify the relationship between Pax, Eya1, Six1 and other genes, and have a strong likelihood of providing significant insight at the molecular level into the early developmental process of otic morphogenesis.